The long-term objective for the research described within this portion of the program project is aimed toward the development of general and comprehensive methodologies for the determination of reaction and substrate specificities for proteins of unknown function. This goal will be pursued by concentrating on the elucidation of the substrate and reaction profiles for the entire ensemble of enzymes within the amidohydrolase superfamily. This superfamily of enzymes has been shown to catalyze the hydrolysis of P-O, P-S, P-F, P-C, C-N, C-O, and C-CI bonds in addition to the cleavage of C-C bonds. The structural hallmark for this superfamily of enzymes is an active site at the C-terminal end of an (beta/alpha)8-barrel protein that contains a mononuclear or binuclear metal center that functions predominantly, but not exclusively, to activate solvent water for nucleophilic attack on electrophilic functional groups. Prominent members of this family of enzymes include dihydroorotase, urease, phosphotriesterase, and adenosine deaminase. The substrate and reaction diversity contained within this enzyme superfamily will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities from existing structural templates. Bioinformatic analyses have identified more than 2,000 members of this superfamily within the 1 million proteins sequenced to date from all organisms. Moreover, in excess of thirty different chemical reactions have been identified thus far for members of this superfamily of enzymes and preliminary evidence suggests that more than 100 additional reactions remain to be elucidated. The specific chemical reactions catalyzed by members of this superfamily with unknown catalytic functions will be experimentally determined by direct biochemical assessment of substrate libraries generated with the aid of high throughput computational docking in conjunction with high resolution x-ray crystallography. These studies will complement the cloning, expression, and purification of specific members of the Superfamily where the biochemical reaction profile is currently unknown. In order to enhance the modeling of the amidohydrolase active sites and docking protocols, representative examples for all of the known isofunctional members of this enzyme superfamily will be structurally characterized by x-ray crystallography. The concerted and synergistic application of biochemical, computational, and structural methodologies described in this application will amplify the evolutionary diversity of the reactions catalyzed by members of the amidohydrolase superfamily and, more importantly, provide general and practical approaches for the assignment of function to proteins of unknown substrate and reaction specificity.